Use of pirenoxine for the protection of corneal tissues in photokeractomy

ABSTRACT

Pirenoxine or 1-hydroxy-5-oxo-5H-pyrido-[3,2a]-phenoxazin-3-carboxylic acid, also called pirfenossone and already used as anti-cataract agent, in the form of a pharmaceutically acceptable salt thereof, if desired, is used for the protection of the corneal tissue in photokeratectomy interventions, as cornea photoablation using excimer laser, for both refractive and therapeutic purpose. In fact pirenoxine is able to inhibit, in the cornea, the oxidative phenomena determined by reactive oxygen species (ROS) which are produced within the tissues following the laser irradiation.

The present invention concerns the use of pirenoxine for the protectionof the corneal tissues in photokeratectomy interventions. Moreparticularly, the invention concerns the use of pirenoxine and saltsthereof as agents able to inhibit within the cornea the oxidativephenomena determined by reactive oxygen species (or ROS, reactive oxygenspecies) which are produced in the tissues following the laserirradiation.

As known, the ophthalmic surgery, and particularly the refractive one,which aims to modify the eye refractive power in order to correct notnegligible visual defects makes use of various, less or moreconsolidated or in evolution techniques, some examples of which areradial keratectomy, epikeratofachia and keratomileusis. In addition tothese, also in the ophthalmology field the use of laser, particularlysolid state laser, (like neodymium:yttrium-aluminiumrnarnet laser, knownas Nd:YAG), and, above all, excimer laser, is remarkably increased.

Excimer laser is a pulse laser which, due to the decay of excited noblegas dimers (excimers obtained from gas mixtures of halogen and noblegases), are able to emit large amounts of energy in form of radiationwithin the range of far ultraviolet (UV-C), in the form of pulse trainshaving predetermined duration, frequency and fluence. Any photon emittedduring the irradiation has enough energy to break the intramolecularbonds of the exposed material, in such a way that the irradiatedmolecules are “broken” in small volatile fragments which are expulsed atsupersonic speed embodying a process known as “photodecomposition”.

In the applications using the excimer laser in corneal surgeryinterventions usually an argon-fluorine laser, emitting radiation with awavelength of 193 nm, which is suitable to carry out highly preciseinterventions with an optimal control on the penetration depth and aminimal thermal or mechanical damage effect on adjacent to exposedtissues, is usually employed. Contrary to other lasers used in clinicalfield, the excimer laser does not emit energy concentrated in a focalpoint but it has a radius with a large cross section which, goingthrough suitable slits, is directed to strike large surface cornea zoneswith an accurate control of the shape and sizes of the exposed zones.The emitted energy is almost totally adsorbed by a surface layer withina thickness of few microns and results, by means of evaporation, inablation at every pulse of cornea layers little more thicker thanmolecular, with a reproducibility not attainable by other techniques.

The excimer laser is widely used for corneal refractive re-modelling intechniques known as photorefractive keratectomy or PRK and LASIK (laserintrastromal keratomileusis), for the correction of various ametropiasamong which the most diffused is myopia. As known, the latter is adefect determined by a cornea curvature higher than required by thelength of ocular bulb, so that light rays from outside are refracted ina such way that, before to reach the retina, they converge in a focalpoint. In this circumstance the use of excimer laser provides thatlayers of corneal tissue, the thickness of which is increasing towardthe centre, be ablated reducing therefore the curvature of the cornea.When the technique is used for the correction of hypermetropia, wherein,on the contrary, the modification to be obtained is an increase of thecornea curvature, the amount of ablated tissue within the periphery ofthe exposed zone is more important than in the centre. Finally for thecorrection of the astigmatism which, as known, is an ametropia caused bycurvature difference in various meridians of the ocular surface, thedepth of the ablation can be asymmetric, depending on the meridian to be“flatted”.

More recently the use of the excimer laser has been suggested for thetherapeutic removal of surface corneal tissues, for the treatment ofvarious corneal irregularities and opacities: like of dystrophic,degenerative, cicatricial or infective type. Such an operation, calledphototherapeutic keratectomy or PTK, has been used, for example, for thetreatment of recurrent corneal erosions, post-operation kerattis,corneal dystrophies as Reis-Buckler dystrophies, corneal opacities orcicatrices caused by Herpes simplex, surface irregularities followingsurgical interventions, for example as outcomes from keratoplasty orrefractive corneal interventions. Contrary to refractivephotokeratectomy PTK aims to eliminate irregularities on the cornealsurface in order to flat the profile thereof and therefore involves theablation of tissue layers with different thickness in the various zonesof treated corneal surface.

Although the above described photokeratectomy interventions appear to bean alternative less traumatic than surgical ophthalmic techniques, therestorative process after the photoablation is not without drawbackswhich are less or more transitory and boring or dis-enabling for thepatient, among which, for example, there are corneal cicatricialproblems, generation of under-epithelial opacities called “haze”, whichdetermine a reduction of visual efficiency resulting from “lightscattering” phenomenon (light diffusion) and, in some circumstances, areduction of refractive values as result of operation. It appears to benot debatable by those skilled in the field that at least partially sucheffects result from the formation of free radicals and, generally,reactive oxygen species, which was detected as side effect of UVirradiation and temperature increase occurring in the involved tissues.

As known the term “reactive oxygen species (or substances)”, or ROS,presently collectively means the free radicals and not radical chemicalspecies which currently take part into oxidative biological processesand whose excess with respect to the natural equilibrium conditions isconsidered to be the base of an ever increasing number of degenerativeand pathological phenomena. Specifically the term ROS comprisessuperoxide anionic radical O₂., hydroxyl radical OH⁻, singlet oxygen ¹O₂and the hydrogen peroxide, H₂O₂, as well as alkoxide RO. and peroxideROO. radicals which are generated from organic molecules during theoxidative processes. The activity of these species exerts, within theorganism, on various cellular components, among which there are a largenumber of structural proteins and enzymes, DNA, RNA and, above all, themembrane lipids.

In fact the lipid peroxidation is the most known mechanism by which ROSexert their degenerative activity on the cellular structures damagingpolyunsaturated fatty acids (PUFA) contained in the cytoplasmicmembranes, often as phospholipid esters. In the initial step of thisprocess the action of a free radical abstracts an hydrogen atom H. fromthe lipid chain, forming a free radical R* which undergoes a molecularrearrangement of the double bonds resulting in a conjugated dieneradical. The latter rapidly reacts with molecular oxygen forming thus alipid peroxide radical ROO., which, being a so strong oxidant to attackanother PUFA, starts the propagation step of the reaction. In such a waya lipid hydroperoxide radical, ROOH; and, correspondingly, another lipidperoxide radical ROO., are formed. Therefore the above described mainbranch of the reaction occurs by means of radical chain attacks to themembrane lipids which are thus transformed step by step in thecorresponding hydroperoxides till to the chain termination by means of afree radical.

Various agents naturally occurring in the cellular tissues can performthe above described action, practically functioning as scavengers orantoxidants. Among these the most known are C (ascorbic acid) and E(alpha tocopherol) vitamins, antioxidant enzymes as superoxide dismutase(SOD), catalase, gluthatione peroxidase and various low molecular weightcompounds, among which gluthatione (GSH), tyrosine, uric acid. Thenatural protection from oxidative stresses performed by thesesubstances, however, can not be enough strong to antagonize thedegradation effect of ROS, in which circumstance the lipid peroxidationcan result in an irreversible damage to the cellular membranes.

It has been also demonstrated that the oxidized forms of transitionmetal ions, as Fe³⁺ and Cu²⁺, in the presence of H₂O₂, can acceleratethe oxidative mechanism by a non enzymatic reaction known as Fentonreaction. In the presence of a reducing agent, as ascorbate, part of theoxidized ions is reduced to the lower oxidation state (for example Fe²+)and the reaction, whose rate depends on the Fe³⁺:Fe²⁺ ratio, proceeds,resulting in the conversion of hydrogen peroxide in hydroxyl ion, OH⁻,plus an hydroxyl radical, OH. The latter represents the most reactiveROS.

Although it is difficult to detect ROS due their reactivity andtherefore their short life times, the formation of free radicals intissues subjected to photoablation using excimer laser has been widelydemonstrated. For example the presence of free radicals in bovinecorneas exposed to irradiation using ArF laser has been revealed by EPRspectroscopy (electron paramagnetic resonance) (R. J. Landry et al.,Laser and Light in Ophthalmol., 6: 87-90, 1994), while measurements oftemperature increase at level of the corneal endothelium and analyticaldeterminations of the reduction of the SOD activity at level of theaqueous humour confirmed the formation of ROS in the cornea of PRKtreated rabbits (K. Bigihan et al., Jpn. J. Ophthalmol., 40, 154-157,1996). The lipid peroxidation has been detected, again in the rabbitcornea, following PTK treatment performed using excimer laser, both byhistochemical test and the analytical detection of the presence ofdegradation products in corneal lipid extracts, particularly conjugateddienes and ketodienes (S. Hayashi et al., British J. Ophthalmol. 81,141-144, 1997).

Further it has been pointed out by EPR spectroscopy the generation offree radicals also when corneal tissues are irradiated using solid stateNd:Yag laser, at a wavelength of 213 nm rather than 193 nm, whichwavelength is typical of the argon-fluorine excimer laser. However inthis case in addition to an oxidative damage comparable to that obtainedusing the excimer laser, it has been also detected a more remarkablecytotoxic effect, somehow dependent on the higher wavelength of theradiation (E. Edigeretal., Lasers Surg. Med., 21:88-93, 1997).

In addition to the effect of the UV radiation on the primary productionof ROS, it has been also observed that the chemiotaxis activity of thusformed lipid hydroperoxides withdraws in situ polymorphonucleated cellsand macrophages which in turn, by producing further ROS, enhance thedamaging action of the radiation inducing a set of cytotoxic effects (H.Goto et al., Curr. Eye Res., 10:1009-1014, 1991).

Although the above reported literature demonstrates the formation offree radicals and reactive oxygen species in the photoablation treatmentand relates this phenomenon to other possible post-operationcomplications it is not considered to be particularly important theprotection of the corneal tissues by administration of exogenous agentshaving ROS antagonizing activity both before and after the operation. Ineffect the currently used pharmacological therapy for thephotokeractectomy treatments is consisting of the topic ocularapplication, after the operation, of antibiotics, with the clear purposeto maintain in aseptic conditions the ocular surface during thecicatrization process, and anti-inflammatory drugs (steroidal or,according to the most recent trends, non steroidal) in order to actagainst the post-operation phlogosis conditions.

Therefore the object of the present invention is to provide the cornealtissues involved in UV irradiation, both before and soon after thetreatment, with an agent suitable to perform a protective activityagainst the cellular damage triggered by the reactive oxygen species andto scavenge the action thereof. Particularly the suggested agent must beeffective to oppose the lipid peroxidation in the corneal cellulartissues.

Within the studies about the effects of ROS and the inhibition of lipidperoxidation by various exogenous molecules having scavenging orantioxidant activity it has been found out that pirenoxine, an activeprinciple already known and used therapeutically on another oculardistrict, the crystalline lens, shows a remarkable activity for theinhibition of lipid peroxidation in the comeal tissues and it istherefore able to perform a protective action against the cellularmodifications resulting form laser irradiation.

Pirenoxine or 1-hydroxy-5-oxo-5H-pyrido-[3,2a]-phenoxazin-3-carboxylicacid (also called pirfenossone) is a known compound having the followingformula:

used in ophthalmology, usually in the form of sodium salt thereof, forthe treatment of the cataract. The latter is an abnormal progressivecondition of the eye crystalline lens characterized by an increasingloss of transparency. As known, the cataracts more often result fromdegenerative modifications, often occurring after 50 year age, whilemore rarely they can result from traumas or poison exposure. Initiallythe vision is hazy, then the brilliant lights dazzle diffusely anddistortion and double vision can develop. At the end, if the cataract isnot treated, anopia occurs. In addition to the surgical treatment, whichbecomes necessary for more advanced degenerative states and involves theablation of the crystalline lens (with or without surgical implantationof an intraocular lens) the cataracts can be treated by the ophthalmictopic administration of pirenoxine in the form of collyrium.

It has been postulated that the ability of pirenoxine to inhibit theformation of lenticular opacities results from at least three differentaction mechanisms: (a) inhibition of the oxidation activity of thequinone molecules on the lenticular proteins, by binding their —SHgroups; (b) activation and normalization of the cation pumping activityperformed by the capsule of the crystalline lens; (c) inhibition of thesorbitol synthesis and reduction of the osmotic damage resulting fromthe storage of this substance (S. Iwata, J. Pharmac. Soc. Jap., 1964;844: 435-440; F. Ikemoto et al., in: Proc. 50^(th) Congr. Pharmacol.Soc. Jap., Kanto Region, 1974: I. Korte et al., Ophthalmic Res., 1979;11: 123-125).

Within the most recent studies about the biological activity ofpirenoxine it has been also found out, and it is the object of theeuropean patent application No. EP 0885612, assigned to the presentApplicant, that this molecule, in addition to the activity in thetreatment of cataract, is has anti-inflammatory properties too. Theseproperties, which have been verified on animal models, embody through anaction mechanism not elucidated in the mentioned patent application,although in the above mentioned patent description it is postulated aninhibiting activity of the oxidative catabolism of arachidonic acid,which results in the production of prostaglandins.

According to the present invention it has been found out, as alreadyreported, that pirenoxine can be advantageously used for the protectionof the corneal tissues during excimer laser treatments because it isactive in inhibiting the lipid peroxidation in the corneal cellulartissues.

It is therefore an object of the present invention the use of1-hydroxy-5-oxo-5H-pyrido-[3,2a]-phenoxazin-3-carboxylic acid(pirenoxine) or a pharmaceutically acceptable salt thereof for theproduction of a topic ophthalmic drug suitable for the protection of thecorneal tissues in photokeratectomy interventions. As already pointedout the suggested drug is designed as inhibitor of the ROS activity(reactive oxygen species) at level of comeal tissues and, particularlyas inhibitor of the lipid peroxidation at level of said tissues.

Use of pirenoxine as pre- and post-operation protective agent findsapplication in any photokeratectomy treatment, being further presumablea wider use in those treatments which presently are more diffused, i.e.corneal photoablation by means excimer laser, both refractive andtherapeutic and, in the first case by means of both PRK and LASIKtechnique.

The ophthalmic preparations of the present invention preferably containthe active principle, i.e. pirenoxine or a pharmaceutically acceptablesalt thereof, in amount from 0,0001% to 0,01 weight %, expressed as freeacid. More conveniently said medicaments contain from 0,001% to 0,005weight % of pirenoxine, expressed as free acid, the optimumconcentration being the same as that presently used for the therapy ofthe cataract, i.e. 0,005 weight %. Most conveniently said pirenoxine isin the form of the sodium salt. When used in the form of collyriumcontaining 0,005 weight % of the active principle, the preparationaccording to the invention can be administered, in order to obtain thedesired effect of ROS inhibition, at a dosage of one-two drops twice orthree times a day, preferably two drops three times a day, beginning atleast one or two days before the operation and continuing, after theoperation, over at least one or two days. Generally the dosage andposology can be widely variable without impairing the whole protectiveeffect against ROSs exerted by the product.

The ophthalmic topic drug containing pirenoxine or a salt thereof canbe, generally, in the same forms as prepared or proposable for the useof the same active principle for the therapy of the cataract orophthalmic inflammation, as described in the above mentioned europeanpatent publication EP-A-0885612. Particularly, the product can be in theform of aqueous solution or suspension for collyrium or in the form ofemulsion, ointment, gel or cream. Preferably the product is administeredas aqueous ophthalmic solution. Because of the instability of the activeprinciple, pirenoxine is normally formulated, in the already usedmedicaments for the treatment of the cataract, as a two componentpreparation wherein a first component comprises freeze-dried pirenoxineand the second component comprises an eye acceptable aqueous carrier ordiluent. The two components are reconstituted before the use and thethus obtained solution can be generally stored at ambient temperaturefor about two weeks without degradation.

Generally the compositions containing pirenoxine or a salt thereofaccording to the invention can be formulated according to the known art,for example according to the teachings suggested by “Remington'sPharmaceutical Sciences Handbook”, Hack Publ. Co., U.S.A. Usually one ormore agents for the regulation of tonicity should be added whereby thesolution has a suitable osmolarity value. Any one of the productsusually used in the art can be used, as, for example, sodium chloride,potassium chloride, mannitol, dextrose, boric acid, propylene glycol.The preparation can also comprise acids or bases as agents for theregulation of pH and/or buffers, as, for example, monosodiumphosphate—disodium phosphate, sodium borate—boric acid or sodiumsuccinate—succinic acid systems. For a good tolerability in the eye thepH should be between 4,5 and 8,5. Furthermore the composition shouldalso comprise preservatives and antimicrobial agents, as benzalkoniumchloride, sodium merthiolate or thimerosal, mehyl-, ethyl- andpropyl-paraben, chlorobutanol, as well as chelating or sequesteringagents as edetates or EDTA. If the product is packaged in unit dosecontainers the presence of preservatives can be avoided but, whenmultiple dose containers are used, for example vials for collyriumcontaining from 5 to 15 ml, the presence of the preservatives isnecessary.

In addition the ophthalmic preparation can comprise further optionalingredients, as thickening agents, anti-oxidants, stabilizers, surfaceactive agents, ecc. Only for exemplary purpose the composition of analready commercially available product designed for the treatment of thecataract is described below. The formulation can be suitable also forthe use of the product as cornea protective agent against free radicalsand ROS.

Some experimental results obtained within the scope of the presentinvention are reported below by way of example together with encloseddrawings, wherein:

FIG. 1 shows the effect of 10⁻⁵ M pirenoxine on fluorescence formationof lipidic soluble substances in rabbit corneas after incubation withf-MLP-stimulated autologous macrophages. Each bar±S.E.M. represents themean value (in brackets the number of processed corneas). *: p<0,01 vscontrol. Control values are significantly higher than basal values(p<0,0002):

FIG. 2 shows the in vitro fluorescence formation of lipidic solublesubstances in UV₃₁₂ irradiated (80 mJ/cm²) epithelial corneal cellsafter incubation in presence and in absence of 10⁻⁵ M pirenoxine. Theresults are mean of 3 experiments;

FIG. 3 shows the ex vivo effects of pirenoxine instillations (60 μlevery hour for 8 hours over 2 days) in the rabbit eyes on conjugateddiene formation in the corneas in vitro submitted to iron-induced lipidperoxidation. Each bar±S.E.M. represents the mean value (in brackets thenumber of processed corneas). (a): expressed by difference between thesample and basal value (without iron-induction: 1.3±0,21nmoles/hemicronea; n 0 8). *: p<0,02 vs control.

EXAMPLE Formulation of Freeze-dried Sodium Pirenoxine

The dry powder component of the product has the following composition,wherein the amounts are given for the reconstitution in a 7 ml solution:

sodium pirenoxine 0.376 mg (equivalent to 0.350 mg of pirenoxine)taurine 34.34 mg

In the preparation taurine and sodium pirenoxine are separatelydissolved in deionized water, the two solutions are sterilized byfiltration and then mixed together and subjected to the freeze-dryingprocess.

polyvinyl alcohol 98 mg succinic acid 2.31 mg sodium succinate .6H₂O89.215 mg sodium chloride 34.3 mg benzalkonium chloride 0.175 mg sodiumedetate 0.89 mg deionized water q.b to 7 ml

In addition to the ingredients mentioned in, the above description saidformulation contains PVA as thickening agent The pH of the solventcomponent is 6. The formulation is prepared by firstly mixing anddissolving in water all the ingredients except benzalkonium chloride.After the complete dissolution of all the products benzalkonium chlorideis added under continued stirring and the mixture is sterilized byfiltration. The pH of the reconstituted product is 6-6.3.

Activity Tests as Inhibitor of the Lipid-peroxidation

In order to evaluate the performance of pirenoxine as protective agentagainst the action of ROS in the corneal tissues and particularlyagainst the lipid peroxidation, the in vitro activity of pirenoxine bothin corneal homogenates in presence of Fe(III)-ascorbic acid oxidizingsystem and on whole cornea subjected to the action of ROS generated fromautologous macrophages was evaluated.

In addition the effects of the UV light on the cornea were carefullyinspected because the comeal tissue is continuously exposed to theexternal environment and therefore to the combined action of oxygen andradiation.

The first experiments carried out by UV₃₁₂ irradiating cornealepithelial cells suggest that also in this case pirenoxine providesantioxidation protection.

The same molecule was assayed for its ex vivo action in the protectionof the cornea against the oxidative in vitro attacks catalyzed by thepresence of iron, as well as against the action of an iron physiologicalcomplex, ferritin, previously UV irradiated and then injected in thecornea stroma. In both case pirenoxine gave successful results.

From the experiments carried out up to now pirenoxine results to be aneffective mean to protect the cornea affected from pathologies generatedby reactive oxygen species.

In Vitro Effect of Pirenoxine on the Action of ROS Induced in Epitheliumand Endothelium Homogenates of Rabbit Corneas

The experimental procedure for the evaluation of the inhibiting actionagainst the lipid-peroxidation exerted by pirenoxine in corneaepithelial and endothelial cells made use of Fe(III)-ascorbic acidsystem to induce the peroxidative phenomenon. The oxidative attack onmembrane lipids was confirmed by the spectrophotometric determinationsof both the conjugated dienes and fluorescent lipid-soluble substanceswhich, as known, are generated by the oxidative degradation of lipidmolecules.

Used experimental procedure included the following steps: a) abstractionof the cornea from the eye of male pigmented rabbits suitably selectedand prepared for the study; b) incubation of the latter in 100 μMphosphate buffer, pH 7,5, in the presence of 1000 U collagenase and 5 μMCaCl₂ for 20 hours at 37° C.; c) centrifugation at 35000 rpm at 0° C.for 10 minutes and washings of the sediment with phosphate buffer; d)homogenization of the cellular sediment in 1 ml of buffer, pH 7,4 (10%w/v); e) incubation of a suitable homogenate aliquot with 10 μM FeCl₃and ascorbic acid in phosphate buffer, pH 7,4, at 27° C. for 30 minutesin presence and in absence of 10⁻⁵ M pirenoxine; f) extraction of thelipid-soluble substances using chloroform/methanol mixture (2:1 v/v).The determination of the conjugated dienes contained in the lipidextract was carried out according to Buege et al. (Methods Enzymol., 52:302-310, 1974), whereas the fluorescent lipid-soluble substances weredetermined according to Fletcher et al. (Anal. Biochem. 52: 1-2, 1973).The test results are reported in the table below.

TABLE 1 Conjugated dienes Fluorescence mmol/hemicornea U/hemicorneaHomogenate 2.93 ± 0.14  4.91 ± 0.11  (basal value) Homogenate + 3.49 ±0.13* 5.89 ± 0.11#  Fe(III) (control) Homogenate + 2.77 ± 0.07* 5.22 ±0.13## Fe(III) + 10⁵ M pirenoxine Each value ± SEM represents the meanvalue of at least 3 (x2) determinations *p < 0.05 and #: p < 0.001 vsrelative basal values; **: p < 0.001 and ##: p < 0.005 vs relativecontrols.

From data reported in the above table it is apparent that pirenoxineexerts a clear inhibiting activity against the lipid-peroxidizing actionof ROS, induced by Fe(III)-ascorbic acid system, as can be deduced fromthe remarkable decrease of the conjugated dienes and significantdecrease of the fluorescent lipid-soluble substances when the above-saidmolecule was present.

In Vitro Protective Effect of Pirenoxine on Corneas Subjected to theAction from ROS Produced from f-MLP-stimulated Autologous RabbitMacrophages

In order to evaluate the inhibition exerted by pirenoxine against theoxidizing activity of macrophage produced ROS at level of the cornea,the following procedure was carried out: (a) broncho-alveolar washing ofthe rabbit to obtain the macrophages; (b) abstraction of the cornea fromthe rabbit eye; (c) incubation of the corneas with 10⁻⁷ M f-MLPstimulated or not stimulated macrophages (800000 cells/well) for twohours at 37° C., 5 % CO₂ in presence and in absence of 10⁻⁵ Mpirenoxine; (d) separation and homogenization of the epithelial andendothelial corneal cells and subsequent determination of fluorescenceas described in b, c, d and f steps of the above methodology.

The results reported in FIG. 1 show that by incubating the whole corneastogether with autologous macrophages in the presence of pirenoxine thelevels of the induced fluorescence result remarkably lower than thecontrols and are comparable to those of normal corneas (basal values).

In Vitro Protective Effect of Direnoxine on the Action of ROS Induced inUVB Irradiated Epithelial Cells

The protective effect of pirenoxine on epithelial comeal cells (SIRC)irradiated for 36″ using UV₃₁₂ light (80 mJ/cm²), according to thefollowing procedure: (a) the comeal cells were plated in 35 mm diameterwells; (b) at 80% confluency the cells were contacted with a medium atlow (0,2%) serum content to inhibit the proliferation thereof during theexperiment (c) the cells were irradiated with UV light in presence andin absence of 10⁻⁵ pirenoxine, incubated at 37° C. for 17 hours andhomogenized in 10 mM f) phosphate buffer, pH 7,4; (d) the lipid-solublefluorescent substances and the proteins contained in suitable homogenatealiquots were determined.

The results reported in FIG. 2 and in table 2 show that pirenoxineexerts a protective effect. In fact the lipid-soluble fluorescentsubstances produced from epithelial corneal cells following the UV₃₁₂irradiation and in presence of said molecule were notably lower (abouttwo and half times) than those produced form cells irradiated in thesame way but not protected by pirenoxine and show fluorescence valuesequal to those of not irradiated cells.

TABLE 2 Proteins, U/mg U/well mg/ml protein Cells 17/12 Control 4.0290.405 9.95 UV312 7.3 0.27 27 UV312 + pir. 3.66 0.403 9.08 Cells 20/12Control 1.61 0.171 9.4 UV312 2.256 0.13 17.35 UV312 + pir. 1.8 0.2457.35 Cells 14/01 Control 4.48 0.444 1.08 UV312 1.02 0.189 5.4 UV312 +pir. 0.92 0.333 2.76 Mean 6.81 Control UV₃₁₂ 16.6 UV₃₁₂ 6.4

Ex Vivo Effect of Pirenoxine on Rabbit Corneas Subjected in Vitro to theAction of ROS

Using the same Fe(III)-ascorbic acid system to induce thelipid-peroxidation as in the first described test, the protective actionof pirenoxine has been evaluated ex vivo according to the followingexperimental procedure: a) the right eye of same type rabbits as in theprevious test was topically treated every hour for 8 hours and over 2days with 2 drops of 0,005% pirenoxine in 0,145 M NaCl (1 drop=30 μl,corresponding to about 1,5 μg), whereas the left eye was treated onlywith saline drops (60 μl); b) at the third day the rabbit was sacrificedby pentobarbital injection (100 mg/kg body weight); c) the corneas,abstracted (115-120 mg), were withdrawn and incubated in 100 μMphosphate buffer, pH 7,5, in the presence of 1000 U collagenase and 5 μMCaCl₂ for 20 hours at 37° C.; afterwards: (d) centrifugation at 3500 rpmat 0° C. for 10 minutes and washing of the sediment with phosphatebuffer; (e) homogenization of cellular sediment in 1 ml of pH 7,5buffer; (f) extraction with chloroform/methanol mixture andspectrophotometric determination of the conjugated dienes. Theexperimental results are reported in FIG. 3 ant in table 3 below:

TABLE 3 (conjugated dienes (mmol/hemicornea)) eyes with salineinstillation 1.85 ± 0.31 eyes with pirenoxine instillation 1.34* ± 0.2 Each value ± SEM represents the mean of at least 3 (x2) determinations*p < 0.05

The values reported in table 3 indicate that pirenoxine, administeredtopically in the rabbit eyes, reaches in the cornea such a concentrationto in vitro contrast the lipid-peroxidizing action of ROS, in fact theformation of conjugated dienes in corneas of eyes (right) subjected to0,005% pirenoxine instillation was lower than that present in eyes(left) treated only by saline.

In Vivo Effect of Pirenoxine on Rabbit Corneas Subjected to, IntrastromaInjection of UV Irradiated Ferritin

The in vivo effect of pirenoxine was evaluated according to thefollowing:

(a) rabbits were anaesthetized by low pentobarbital doses (20 mg/kg);(b) 25 μl of 50 μM ferritin in 0,15 M NaCl were injected in the stromaof the cornea by a 0,33×13 mm/29G insulin syringe whereas the controlswere treated with 25 μl physiological solution; (c) in the eyes everyhour two drops (1 drop=30 μl) of 0,005% pirenoxine in 0,145 M NaCl 8times a day over 4 days were instilled, whereas the controls weretreated only with the solvent, in the same amount and frequency; (d) atthe 5^(th) day the animals were sacrificed using a pentobarbitaloverdose (100 mg/kg); (e) the corneas were abstracted and the tissuecells separated and collected according to the procedure described inthe ex vivo experiment; (f) the conjugated dienes and fluorescentsoluble lipid contained in suitable homogenate aliquots were determined.

The obtained data, reported in table 4, point out a reduction of thelipid peroxidation in the corneas of pirenoxine treated eyes, asindicated by the decrease of conjugated dienes and fluorescent lipidsoluble substances.

TABLE 4 Eyes with ferritin Conjugated diene Fluorescence intrastromalinjection Nmoles/hemicornea Unit/hemicornea Eyes with salineinstallation 1.6 4.5 (without ferritin: basal value) Eyes with salineinstallation 2.1 11.5 (control) Eyes with 0.005% 1.7 4.4 pirelnoxineinstillation

In vivo conjugated diene and lipid soluble fluorescence substanceformation in corneas 5 days after the rabbit eyes were submitted tointrastromal injection of UV-irradiated ferritin and topicalinstillation of pirenoxine solution (2 drops every hour for 8 hr over 4days)

The present invention was described with reference to some specificembodiments thereof but it is understood that modifications orvariations can be carried out by those skilled in the art withoutdeparting from the scope thereof.

What is claimed is:
 1. A method for the protection of corneal tissue ina photokeratectomy intervention comprising the administration onto thecorneal tissue of a topical opthalmic drug comprising1-hydroxy-5-oxo-5H-pyrido-phenoxazin-3-carboxylic acid (pirenoxine) or apharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the topical opthalmic drug is an inhibitor of ROS action at thelevel of corneal tissues.
 3. The method of claim 2, wherein the topicalopthalmic drug is an inhibitor of the lipid peroxidation at the corneallevel.
 4. The method of claim 1, wherein the photokeratectomyintervention is a corneal photoblation intervention using an excimerlaser.
 5. The method of claim 1, wherein the topical opthalmic drugcontains from 0.0001% to 0.01% by weight pirenoxine, expressed as freeacid.
 6. The method of claim 5, wherein the topical opthalmic drugcontains from 0.001% to 0.005% by weight pirenoxine, expressed as freeacid.
 7. The method of claim 1, wherein the pirenoxine is in the form ofthe corresponding sodium salt.
 8. The method of claim 1, wherein thetopical opthalmic drug is in the form of an aqueous solution orsuspension for collyrium or in the form of an emulsion, an ointment, agel, or a cream.
 9. The method of claim 8, wherein the aqueous solutionis obtained by reconstitution of a two component preparation, wherein afirst component comprises freeze-dried pirenoxine, in the form a sodiumsalt, together with an eye acceptable carrier, and the second componentcomprises an eye acceptable aqueous carrier or diluent.